Total internal reflection fluorescence (TIRF) microscopy is a commercially available technology for detection and visual monitoring of fluorescently labeled nano- and microscopic objects, such as cells, nanoparticles, lipid vesicles, molecules etc., in the close vicinity of or on surfaces.
In cell and molecular biology, different molecular events occurring in or close to cellular surfaces such as cell adhesion, binding to cells of hormones, secretion of neurotransmitters, membrane dynamics and cellular interaction with inorganic surfaces have been studied with conventional fluorescence microscopes. However, fluorophores that are bound to the specimen and those in the surrounding medium often exist in an equilibrium state. When these molecules are excited and detected with a conventional fluorescence microscope, the resulting fluorescence from those fluorophores bound to the cellular and/or inorganic surface is often overwhelmed by the background fluorescence due to the much larger number of non-bound molecules.
The concept of using total internal reflection to illuminate cells or other small-scale fluorescently labelled objects contacting the surface of a transparent material such as e.g. glass has been known for several decades. A TIRF microscope uses an evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to a glass-water interface. The evanescent wave is generated only when the incident light is totally internally reflected at the glass-water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium. Thus the TIRF microscope enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells. TIRF can also be used to observe the fluorescence of single fluorescent nanoscale objects as well as single molecules, making it an important tool of biophysics and quantitative biology.
Examples of TIRF-related techniques are given in U.S. Pat. No. 6,753,188 and WO2007/077218.
A potential drawback with the TIRF technology is that it typically requires labelling of molecules under investigation, which may affect the material, e.g. live cells or nanoparticles, to be studied. Furthermore, from a practical point of view, fluorescence labelling is not always straight forward to implement.
It is thus of interest to provide a technique capable of doing in principle the same thing as TIRF but that does not require labelling. It is also of interest to provide a technique that is more sensitive than TIRF, without being too complicated and costly.